Atrial-selective targeting of arrhythmogenic phase-3 early afterdepolarizations in human myocytes.
ABSTRACT: We have previously shown that non-equilibrium Na(+) current (INa) reactivation drives isoproterenol-induced phase-3 early afterdepolarizations (EADs) in mouse ventricular myocytes. In these cells, EAD initiation occurs secondary to potentiated sarcoplasmic reticulum Ca(2+) release and enhanced Na(+)/Ca(2+) exchange (NCX). This can be abolished by tetrodotoxin-blockade of INa, but not ranolazine, which selectively inhibits ventricular late INa.Since repolarization of human atrial myocytes is similar to mouse ventricular myocytes in that it is relatively rapid and potently modulated by Ca(2+), we investigated whether similar mechanisms can evoke EADs in human atrium. Indeed, phase-3 EADs have been shown to re-initiate atrial fibrillation (AF) during autonomic stimulation, which is a well-recognized initiator of AF.We integrated a Markov model of INa gating in our human atrial myocyte model. To simulate experimental results, we rapidly paced this cell model at 10Hz in the presence of 0.1?M acetylcholine and 1?M isoproterenol, and assessed EAD occurrence upon return to sinus rhythm (1Hz).Cellular Ca(2+) loading during fast pacing results in a transient period of hypercontractility after return to sinus rhythm. Here, fast repolarization and enhanced NCX facilitate INa reactivation via the canonical gating mode (i.e., not late INa burst mode), which drives EAD initiation. Simulating ranolazine administration reduces atrial peak INa and leads to faster repolarization, during which INa fails to reactivate and EADs are prevented.Non-equilibrium INa reactivation can critically contribute to arrhythmias, specifically in human atrial myocytes. Ranolazine might be beneficial in this context by blocking peak (not late) atrial INa.
Project description:Early afterdepolarizations (EADs) are triggers of cardiac arrhythmia driven by L-type Ca(2+) current (ICaL) reactivation or sarcoplasmic reticulum Ca(2+) release and Na(+)/Ca(2+) exchange. In large mammals the positive action potential plateau promotes ICaL reactivation, and the current paradigm holds that cardiac EAD dynamics are dominated by interaction between ICaL and the repolarizing K(+) currents. However, EADs are also frequent in the rapidly repolarizing mouse action potential, which should not readily permit ICaL reactivation. This suggests that murine EADs exhibit unique dynamics, which are key for interpreting arrhythmia mechanisms in this ubiquitous model organism. We investigated these dynamics in myocytes from arrhythmia-susceptible calcium calmodulin-dependent protein kinase II delta C (CaMKII?C)-overexpressing mice (Tg), and via computational simulations.In Tg myocytes, ?-adrenergic challenge slowed late repolarization, potentiated sarcoplasmic reticulum Ca(2+) release, and initiated EADs below the ICaL activation range (-47 ± 0.7 mV). These EADs were abolished by caffeine and tetrodotoxin (but not ranolazine), suggesting that sarcoplasmic reticulum Ca(2+) release and Na(+) current (INa), but not late INa, are required for EAD initiation. Simulations suggest that potentiated sarcoplasmic reticulum Ca(2+) release and Na(+)/Ca(2+) exchange shape late action potential repolarization to favor nonequilibrium reactivation of INa and thereby drive the EAD upstroke. Action potential clamp experiments suggest that lidocaine eliminates virtually all inward current elicited by EADs, and that this effect occurs at concentrations (40-60 ?mol/L) for which lidocaine remains specific for inactivated Na(+) channels. This strongly suggests that previously inactive channels are recruited during the EAD upstroke, and that nonequilibrium INa dynamics underlie murine EADs.Nonequilibrium reactivation of INa drives murine EADs.
Project description:Most cardiac arrhythmias occur intermittently. As a cellular precursor of lethal cardiac arrhythmias, early afterdepolarizations (EADs) during action potentials(APs) have been extensively investigated, and mechanisms for the occurrence of EADs on a beat-to-beat basis have been proposed. However, no previous study explains slow fluctuations in EADs, which may underlie intermittency of EAD trains and consequent arrhythmias. We hypothesize that the feedback of intracellular calcium and sodium concentrations ([Na](i) and [Ca](i)) that influence membrane voltage (V) can explain EAD intermittency.AP recordings in rabbit ventricular myocytes revealed intermittent EADs, with slow fluctuations between runs of APs with EADs present or absent. We then used dynamical systems analysis and detailed mathematical models of rabbit ventricular myocytes that replicate the observed behavior and investigated the underlying mechanism. We found that a dominance of inward Na-Ca exchanger current (I(NCX)) over Ca-dependent inactivation of L-type Ca current (I(CaL)) forms a positive feedback between [Ca](i) and V, thus resulting in 2 stable AP states, with and without EADs (ie, bistability). Slow changes in [Na](i) determine the transition between these 2 states, forming a bistable on-off switch of EADs. Tissue simulations showed that this bistable switch of cellular EADs provided both a trigger and a functional substrate for intermittent arrhythmias in homogeneous tissues.Our study demonstrates that the interaction among V, [Ca](i), and [Na](i) causes slow on-off switching (or bistability) of AP duration in cardiac myocytes and EAD-mediated arrhythmias and suggests a novel possible mechanism for intermittency of cardiac arrhythmias.
Project description:Class 1 antiarrhythmic drugs are highly effective in restoring and maintaining sinus rhythm in atrial fibrillation patients but carry a risk of ventricular tachyarrhythmia. The antianginal agent ranolazine is a prototypic atrial-selective voltage-gated Na+ channel blocker but the mechanisms underlying its atrial-selective action remain unclear.The present study examined the mechanisms underlying the atrial-selective action of ranolazine.Whole-cell voltage-gated Na+ currents (INa) were recorded at room temperature (?22°C) from rabbit isolated left atrial and right ventricular myocytes.INa conductance density was ?1.8-fold greater in atrial than in ventricular cells. Atrial INa was activated at command potentials ?7 mV more negative and inactivated at conditioning potentials ?11 mV more negative than ventricular INa. The onset of inactivation of INa was faster in atrial cells than in ventricular myocytes. Ranolazine (30 ?M) inhibited INa in atrial and ventricular myocytes in a use-dependent manner consistent with preferential activated/inactivated state block. Ranolazine caused a significantly greater negative shift in voltage of half-maximal inactivation in atrial cells than in ventricular cells, the recovery from inactivation of INa was slowed by ranolazine to a greater extent in atrial myocytes than in ventricular cells, and ranolazine produced an instantaneous block that showed marked voltage dependence in atrial cells.Differences exist between rabbit atrial and ventricular myocytes in the biophysical properties of INa. The more negative voltage dependence of INa activation and inactivation, together with trapping of the drug in the inactivated channel, underlies an atrial-selective action of ranolazine.
Project description:PURPOSE: Torsades de pointes (TdP) tachycardias are triggered, polymorphic ventricular arrhythmias arising from early afterdepolarizations (EADs) and increased dispersion of repolarization. Ranolazine is a new agent which reduces pathologically elevated late INa but also IKr . Aim of this study was to evaluate the effects of ranolazine in a validated isolated Langendorff-perfused rabbit heart model. METHODS: TdP was reproducibly induced with d-sotalol (10(-4) mol/L) and low potassium (K) (1.0 mmol/L for 5 min, pacing at CL 1000 ms). In 10 hearts, ECG and 8 epi- and endocardial monophasic action potentials were recorded. Action potential duration (APD) was measured at 90% repolarization and dispersion defined as APD max-min. RESULTS: D-sotalol prolonged APD90 and increased dispersion of APD90 , simultaneously causing EADs and induction of TdP. The combination of d-sotalol and two concentrations of ranolazine did not increase dispersion of ventricular APD90 as compared to vehicle. Ranolazine at 5 μmol/L did not cause additional induction of EADs and/or TdP but also did not significantly suppress arrhythmogenic triggers. The higher concentration of ranolazine (10 μmol/L) in combination with d-sotalol caused further prolongation of APD90 , at the same time reduction in APD90 dispersion. In parallel, the incidence of EADs was reduced and an antitorsadogenic effect was seen. CONCLUSIONS: In the healthy isolated rabbit heart (where late INa is not elevated), ranolazine does not cause proarrhythmia but exerts antiarrhythmic effects in a dose-dependent manner against d-sotalol/low K-induced TdP. This finding-despite additional APD prolongation-supports the safety of a combined use of both drugs and merits clinical investigation.
Project description:<h4>Aims</h4>The transient outward potassium current (I(to)) plays important roles in action potential (AP) morphology and dynamics; however, its role in the genesis of early afterdepolarizations (EADs) is not well understood. We aimed to study the effects and mechanisms of I(to) on EAD genesis in cardiac cells using combined experimental and computational approaches.<h4>Methods and results</h4>We first carried out patch-clamp experiments in isolated rabbit ventricular myocytes exposed to H(2)O(2) (0.2 or 1 mM), in which EADs were induced at a slow pacing rate. EADs were eliminated by either increasing the pacing rate or blocking I(to) with 2 mM 4-aminopyridine. In addition to enhancing the L-type calcium current (I(Ca,L)) and the late sodium current, H(2)O(2) also increased the conductance, slowed inactivation, and accelerated recovery from the inactivation of I(to). Computer simulations showed that I(to) promoted EADs under the condition of reduced repolarization reserve, consistent with the experimental observations. However, EADs were only promoted in the intermediate ranges of the I(to) conductance and the inactivation time constant. The underlying mechanism is that I(to) lowers the AP plateau voltage into the range at which the time-dependent potassium current (namely I(Ks)) activation is further slowed and I(Ca,L) is available for reactivation, leading to voltage oscillations to manifest EADs. Further experimental studies in cardiac cells of other species validated the theoretical predictions.<h4>Conclusion</h4>In cardiac cells, I(to), with a proper conductance and inactivation speed, potentiates EADs by setting the AP plateau into the voltage range where I(Ca,L) reactivation is facilitated and I(Ks) activation is slowed.
Project description:Hypokalemia is known to promote ventricular arrhythmias, especially in combination with class III antiarrhythmic drugs like dofetilide. Here, we evaluated the underlying molecular mechanisms.Arrhythmias were recorded in isolated rabbit and rat hearts or patch-clamped ventricular myocytes exposed to hypokalemia (1.0-3.5 mmol/L) in the absence or presence of dofetilide (1 ?mol/L). Spontaneous early afterdepolarizations (EADs) and ventricular tachycardia/fibrillation occurred in 50% of hearts at 2.7 mmol/L [K] in the absence of dofetilide and 3.3 mmol/L [K] in its presence. Pretreatment with the Ca-calmodulin kinase II (CaMKII) inhibitor KN-93, but not its inactive analogue KN-92, abolished EADs and hypokalemia-induced ventricular tachycardia/fibrillation, as did the selective late Na current (INa) blocker GS-967. In intact hearts, moderate hypokalemia (2.7 mmol/L) significantly increased tissue CaMKII activity. Computer modeling revealed that EAD generation by hypokalemia (with or without dofetilide) required Na-K pump inhibition to induce intracellular Na and Ca overload with consequent CaMKII activation enhancing late INa and the L-type Ca current. K current suppression by hypokalemia and dofetilide alone in the absence of CaMKII activation were ineffective at causing EADs.We conclude that Na-K pump inhibition by even moderate hypokalemia plays a critical role in promoting EAD-mediated arrhythmias by inducing a positive feedback cycle activating CaMKII and enhancing late INa. Class III antiarrhythmic drugs like dofetilide sensitize the heart to this positive feedback loop.
Project description:Early afterdepolarizations (EADs) are spontaneous depolarizations during the repolarization phase of an action potential in cardiac myocytes. It is widely known that EADs are promoted by increasing inward currents and/or decreasing outward currents, a condition called reduced repolarization reserve. Recent studies based on bifurcation theories show that EADs are caused by a dual Hopf-homoclinic bifurcation, bringing in further mechanistic insights into the genesis and dynamics of EADs. In this study, we investigated the EAD properties, such as the EAD amplitude, the inter-EAD interval, and the latency of the first EAD, and their major determinants. We first made predictions based on the bifurcation theory and then validated them in physiologically more detailed action potential models. These properties were investigated by varying one parameter at a time or using parameter sets randomly drawn from assigned intervals. The theoretical and simulation results were compared with experimental data from the literature. Our major findings are that the EAD amplitude and takeoff potential exhibit a negative linear correlation; the inter-EAD interval is insensitive to the maximum ionic current conductance but mainly determined by the kinetics of ICa,L and the dual Hopf-homoclinic bifurcation; and both inter-EAD interval and latency vary largely from model to model. Most of the model results generally agree with experimental observations in isolated ventricular myocytes. However, a major discrepancy between modeling results and experimental observations is that the inter-EAD intervals observed in experiments are mainly between 200 and 500 ms, irrespective of species, while those of the mathematical models exhibit a much wider range with some models exhibiting inter-EAD intervals less than 100 ms. Our simulations show that the cause of this discrepancy is likely due to the difference in ICa,L recovery properties in different mathematical models, which needs to be addressed in future action potential model development.
Project description:Early afterdepolarization (EAD) is known to cause lethal ventricular arrhythmias in long QT syndrome (LQTS). In this study, dynamical mechanisms of EAD formation in human ventricular myocytes (HVMs) were investigated using the mathematical model developed by ten Tusscher and Panfilov (Am J Physiol Heart Circ Physiol 291, 2006). We explored how the rapid (IKr) and slow (IKs) components of delayed-rectifier K+ channel currents, L-type Ca2+ channel current (ICa L), Na+/Ca2+ exchanger current (INCX), and intracellular Ca2+ handling via the sarcoplasmic reticulum (SR) contribute to initiation, termination and modulation of phase-2 EADs during pacing in relation to bifurcation phenomena in non-paced model cells. Parameter-dependent dynamical behaviors of the non-paced model cell were determined by calculating stabilities of equilibrium points (EPs) and limit cycles, and bifurcation points to construct bifurcation diagrams. Action potentials (APs) and EADs during pacing were reproduced by numerical simulations for constructing phase diagrams of the paced model cell dynamics. Results are summarized as follows: (1) A modified version of the ten Tusscher-Panfilov model with accelerated ICaL inactivation could reproduce bradycardia-related EADs in LQTS type 2 and ?-adrenergic stimulation-induced EADs in LQTS type 1. (2) Two types of EADs with different initiation mechanisms, ICaL reactivation-dependent and spontaneous SR Ca2+ release-mediated EADs, were detected. (3) Termination of EADs (AP repolarization) during pacing depended on the slow activation of IKs. (4) Spontaneous SR Ca2+ releases occurred at higher Ca2+ uptake rates, attributable to the instability of steady-state intracellular Ca2+ concentrations. Dynamical mechanisms of EAD formation and termination in the paced model cell are closely related to stability changes (bifurcations) in dynamical behaviors of the non-paced model cell, but they are model-dependent. Nevertheless, the modified ten Tusscher-Panfilov model would be useful for systematically investigating possible dynamical mechanisms of EAD-related arrhythmias in LQTS.
Project description:Early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) are voltage oscillations known to cause cardiac arrhythmias. EADs are mainly driven by voltage oscillations in the repolarizing phase of the action potential (AP), while DADs are driven by spontaneous calcium (Ca) release during diastole. Because voltage and Ca are bidirectionally coupled, they modulate each other's behaviors, and new AP and Ca cycling dynamics can emerge from this coupling. In this study, we performed computer simulations using an AP model with detailed spatiotemporal Ca cycling incorporating stochastic openings of Ca channels and ryanodine receptors to investigate the effects of Ca-voltage coupling on EAD and DAD dynamics. Simulations were complemented by experiments in mouse ventricular myocytes. We show that: 1) alteration of the Ca transient due to increased ryanodine receptor leakiness and/or sarco/endoplasmic reticulum Ca ATPase activity can either promote or suppress EADs due to the complex effects of Ca on ionic current properties; 2) spontaneous Ca waves also exhibit complex effects on EADs, but cannot induce EADs of significant amplitude without the participation of ICa,L; 3) lengthening AP duration and the occurrence of EADs promote DADs by increasing intracellular Ca loading, and two mechanisms of DADs are identified, i.e., Ca-wave-dependent and Ca-wave-independent; and 4) Ca-voltage coupling promotes complex EAD patterns such as EAD alternans that are not observed for solely voltage-driven EADs. In conclusion, Ca-voltage coupling combined with the nonlinear dynamical behaviors of voltage and Ca cycling play a key role in generating complex EAD and DAD dynamics observed experimentally in cardiac myocytes, whose mechanisms are complex but analyzable.
Project description:In congenital and acquired long QT type 2, women are more vulnerable than men to torsade de pointes. In prepubertal rabbits (and children), the arrhythmia phenotype is reversed; however, females still have longer action potential durations than males. Thus, sex differences in K(+) channels and action potential durations alone cannot account for sex-dependent arrhythmia phenotypes. The L-type calcium current (I(Ca,L)) is another determinant of action potential duration, Ca(2+) overload, early afterdepolarizations (EADs), and torsade de pointes. Therefore, sex, age, and regional differences in I(Ca,L) density and in EAD susceptibility were analyzed in epicardial left ventricular myocytes isolated from the apex and base of prepubertal and adult rabbit hearts. In prepubertal rabbits, peak I(Ca,L) at the base was 22% higher in males than females (6.4+/-0.5 versus 5.0+/-0.2 pA/pF; P<0.03) and higher than at the apex (6.4+/-0.5 versus 5.0+/-0.3 pA/pF; P<0.02). Sex differences were reversed in adults: I(Ca,L) at the base was 32% higher in females than males (9.5+/-0.7 versus 6.4+/-0.6 pA/pF; P<0.002) and 28% higher than the apex (9.5+/-0.7 versus 6.9+/-0.5 pA/pF; P<0.01). Apex-base differences in I(Ca,L) were not significant in adult male and prepubertal female hearts. Western blot analysis showed that Ca(v)1.2alpha levels varied with sex, maturity, and apex-base, with differences similar to variations in I(Ca,L); optical mapping revealed that the earliest EADs fired at the base. Single myocyte experiments and Luo-Rudy simulations concur that I(Ca,L) elevation promotes EADs and is an important determinant of long QT type 2 arrhythmia phenotype, most likely by reducing repolarization reserve and by enhancing Ca(2+) overload and the propensity for I(Ca,L) reactivation.